Hiptage yangshuoensis (Malpighiaceae), a new species on karst hills close to Lijiang River, Guangxi, China, based on molecular and morphological data

Abstract Hiptage yangshuoensis K.Tan & K.S.Nguyen, a new species of Hiptage collected from a karst cliff close to the Lijiang River, Northeast of Guangxi Zhuang Autonomous Region, China, is described and illustrated based on molecular and morphological data. Hiptage yangshuoensis shares some morphological similarities with the H. multiflora F.N.Wei, but easily distinguished by its long pedicels with articulate at top, one large calyx gland, oblanceolate middle wing and lanceolate lateral wings of samara, and young branch covered rusty sericeous. The new species status is also supported by molecular phylogenetic analyses based on nuclear ribosome internal transcribed spacer (nrITS), which showed distinct systematic distinctiveness from the most morphologically similar species, H. multiflora.

usually with two basal glands on abaxial surface; stipules minute, glandlike, or absent.Flowers white, sometimes pinkish, fragrant, bisexual, zygomorphic.The style is deflected away from the floral axis, either to the left in left-styled flowers or to the right in rightstyled flowers, and characterizes these as "mirror-image flowers" (Ren et al., 2013).Stamens are 10 in number and unequal; one, much larger than the others, acts as the "pollinating anther" and serves reproductive functions, while another serves as the "feeding anther," providing rewards to pollinators (Ren et al., 2013).Samaras usually have 3 wings; the abaxial wing is long and erect, whereas the other 2 lateral wings are short and spread outward (Chen & Funston, 2008;Sirirugsa, 1991).In recent years, five new species of Hiptage have been described in Southwestern China (Dong et al., 2020;Tan et al., 2019;Yang et al., 2018;Zhang et al., 2023).
Current molecular evidence suggests that the Hiptage is monophyletic, with Chlorohiptage T.V. Do, T.A.Le & R.F.Almeida from Vietnam identified as its sister group (Do et al., 2024).Although there is only a single marker (ITS) phylogenetic study of Hiptage and many clades with weak support, it is clear that H. stellulifera Arènes is the most basal species within the genus (Almeida & van den Berg, 2022;Tan et al., 2019).| 3 of 7 WANG et al.Funston, 2008).However, it differs in the lanceolate lateral wings of samara, young branch covered rusty sericeous, and pedicel articulate at top, both of which are critical morphological traits for species taxonomy in Hiptage (Chen & Funston, 2008;Ren, 2015).Therefore, we describe it as a new species, depicted and illustrated here.

| Taxonomy
Morphological analysis of the new Hiptage species was conducted using fresh or preserved specimens, with measurements of specific traits averaged across five individuals.In-field photographs captured the plants' and its flowers' characteristics (Figure 1).Comparative morphological evaluations were conducted with type specimens of Hiptage from herbaria (IBK, KUN, IBSC, and HUTB), utilizing protologues and herbarium samples alongside digital resources from JSTOR Global Plants (http:// plants.jstor.org) and the Chinese Virtual Herbarium (http:// www.cvh.ac.cn), as detailed in Table S1, and relevant taxonomic literature (e.g., Tan et al., 2019;Yang et al., 2018;Zhang et al., 2023).The morphological terminology follows Niedenzu (1924), Jacobs (1955), Anderson et al. (2006), Chen andFunston (2008), andAlmeida andMorais (2022).The conservation status assessments of the new species H. yangshuoensis were based on the International Union for Conservation of Nature guidelines (IUCN Standards and Petitions Committee, 2024).

| Phylogenetic analysis
To verify the taxonomic status of the new species within Hiptage, we conducted a phylogenetic analysis using the nuclear riboso-   sequence data for 39 samples from GenBank (Table S2)  (Table S2).Dried leaf material of the proposed new species was collected from the type locality in Fuli Town, Yangshuo County (Guangxi, China).Two samples from the new species and one sample from H. multiflora were newly sequenced in this study to provide molecular evidence (Table S2).Total genomic DNA was isolated from dried leaf samples using a CTAB protocol adapted from Doyle and Doyle (1987).ITS fragment sequencing involved bidirectional evaluation using PhyDE for accuracy (Müller et al., 2010), followed by manual alignment in MEGA v.7 (Kumar et al., 2016).Using ModelFinder (Kalyaanamoorthy et al., 2017), the SYM + G4 model and the SYM + R2 model were selected as the optimal substitution models for maximum likelihood (ML) analysis and Bayesian inference (BI), respectively.

| RE SULTS AND D ISCUSS I ON
The  Hiptage yangshuoensis is morphologically similar to H. multiflora.
However, the new species differs by several key morphological characters (summarized in Table 1) and molecular data (Figure 2).
Moreover, the H. yangshuoensis is found in northeastern Guangxi (Figure 4), whereas H. multiflora inhabits the southwestern region of Guangxi, with over 500 kilometers separating their habitats.This significant distance, along with the distinctive "terrestrial islands" ecosystem created by the karst landscape, readily elucidates the speciation phenomenon observed within these karst species (e.g., Ke et al., 2022;Wang et al., 2017).Presently, this new species marks another recent discovery at the northern boundary of the genus's distribution range, succeeding the identification of several Hiptage species in northwestern Yunnan (Dong et al., 2020;Tan et al., 2019;Zhang et al., 2023).Diagnosis.Hiptage yangshuoensis differs from the closely related H. multiflora by the rusty sericeous (vs.white to rusty puberulent) young branch, pedicels 1.5-2.3cm and articulate at top (vs.ca. 1 cm, at middle), 1 calyx gland (vs.initially 2, the calyx glands merge at the base into one as they mature), and samara with oblanceolate (vs.obovate) middle wing and lanceolate (vs. oblong) lateral wing.
During our biodiversity surveying along the Lijiang River, Guilin City, Northeast of Guangxi Zhuang Autonomous Region, China, we collected specimens from a distinct morphotype of Hiptage growing on a karst cliff close to the Lijiang River.After detailed analyses of morphological characters and molecular data, along with comparisons of specimens, we found that the species of our collection is most morphologically similar to H. multiflora F.N.Wei (Chen & F I G U R E 1 Hiptage yangshuoensis (a) habitat, (b) habit, (c) inflorescence, (d) flower in side view, (e) flower in frontal view, (f) petals, (g) samara, (h) fruiting branch, (i) branchlets, (j) calyx gland (showing articulate at top of pedicel).
mal internal transcribed spacer (ITS) region.We downloaded ITS F I G U R E 2 Molecular phylogenetic tree of Hiptage based on ITS sequences.Bayesian posterior probability (PP) and ML bootstrap values (BS) are shown above branches as PP/BS (only shown if PP > 0.5 and BS > 50).

F
Hiptage multiflora (a) branchlets, (b) young branch covered white to rusty sericeous, (c) leaf glands, (d) flower in side view, (e) samara, (f) calyx glands initially 2, merge at the base into one as they mature.The arrow indicates the articulate position on the pedicel.

4
Figure 1 Type.CHINA.Guangxi Zhuang Autonomous Region: Guilin City, Yangshuo County, Fuli Town, close to Lijiang River, 24°46′41″ N, 110°33′11″ E, 120 m a.s.l., 11 Apr.2023, K. Tan23tk042201 (Holotype: IBK!; Isotype: IBK!; Paratype: IBK! IBK00414206).Description.Woody shrubs; young branches rusty sericeous, hairs adpressed, older twigs glabrous, with white or greenish lenticels, rounded, coarse warts dotted.Leaves opposite; petiole ca.0.5 cm long with yellowish brown eglandular sericeous; leaf blades ovate, 7.0-16.0× 2.5-6.0 cm, coriaceous; densely rusty sericeous on abaxial surfaces when young, and glabrous at mature, base obtuse or broadly cuneate, margin entire or slightly undulate, apex acute or acuminate, abaxially often with 2 marginal glands near the base; lateral veins 5-8 pairs, prominent on both surfaces.Thyrses, terminal or axillary; main axis 3.5-6.0cm long, rusty F I G U R E 4 Distribution map showcasing the new species, Hiptage yangshuoensis, alongside its similar species, H. multiflora.[Correction added September 03 2024, after first online publication: Figure 4 has been updated.] Habitat and distribution.Hiptage yangshuoensis is only known from the karst hill close to Lijiang River, at an elevation ca.120 m, near Yangshuo County, northeast of Guangxi Zhuang Autonomous Region, China (Figure 4).Conservation status.Since the only known population of Hiptage yangshuoensis is growing on a karst cliff close to the Lijiang River in the northeast Guangxi Zhuang Autonomous Region, we have not discovered the wild population outside of the abovementioned place, information known about the population status and natural distribution range of the new species is very limited.Currently, only about 10 individuals have been identified on the karst cliff by unmanned aerial vehicle (UAV).Therefore, we suggest that the new species H. yangshuoensis should be considered "Critically Endangered" [CR, B2a,b (iii, iv, v)] according to current IUCN Red List Categories and Criteria (IUCN Standards and Petitions Committee, 2024).Methodology (equal); software (equal); writingoriginal draft (supporting).Yao Ning: Data curation (supporting); formal analysis (equal); software (equal).Xiao-Juan Li: Formal analysis (equal); methodology (equal).Ke Tan: Conceptualization (lead); resources (lead); writing -original draft (lead); writing -review and editing (equal).Khang Sinh Nguyen: Data curation (lead); writingreview and editing (equal).
Morphological comparison of key characteristics in H. yangshuoensis and H. multiflora.-2.3 cm long, articulate at top, green, rusty sericeous ca. 1 cm long, articulate at middle, red, white puberulent aligned matrix of ITS sequences consisted of 687 bp, of which 466 sites were identical, 123 (17.9%) were parsimony informative, and 98 parsimony-uninformative variable characters.The phylogenetic analysis showed that Hiptage is a monophyletic group (PP/BS = 1/100), with H. stellulifera (PP/BS = 1/100) being the first lineage to diverge, consistent with previous studies (Almeida & van den Berg, 2022; Tan 1 sepal gland, and sepal gland not decurrent to pedicel.However, H. yangshuoensis is distinguished by its samara middle wing oblanceolate and lateral wing lanceolate (vs.obovate and oblong), young branch covered rusty sericeous (vs.white to rusty puberulent), and pedicel articulate at top (vs.atmiddle).Other features, like pedicels, differentiate the two species, as detailed in Table1 and Figure 3.TA B L E 1